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Characterizing the distribution and rates of microbial sulfate reduction at Middle Valley
hydrothermal vents
Kiana L. Frank1, Daniel R. Rogers1, Heather C. Olins1, Charles Vidoudez1 and Peter R. Girguis1*
1 = Department of Organismic and Evolutionary Biology, Harvard University, 16 Divinity Avenue,
Cambridge, MA 02138-2020.
* = send correspondence to pgirguis@oeb.harvard.edu
Running Title: Sulfate reduction rates at hydrothermal vents
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ABSTRACT
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Few studies have directly measured sulfate reduction at hydrothermal vents, and
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relatively little is known about how environmental or ecological factors influence rates of
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sulfate reduction in vent environments. A better understanding of microbially mediated sulfate
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reduction in hydrothermal vent ecosystems may be achieved by integrating ecological and
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geochemical data with metabolic rate measurements. Here we present rates of microbially
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mediated sulfate reduction from three distinct hydrothermal vents in the Middle Valley vent
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field along the Juan de Fuca Ridge, as well as assessments of bacterial and archaeal diversity,
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estimates of total biomass and the abundance of functional genes related to sulfate reduction,
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and in situ geochemistry. Maximum rates of sulfate reduction occurred at 90°C in all three
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deposits. Pyrosequencing and functional gene abundance data reveal differences in both
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biomass and community composition among sites, including differences in the abundance of
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known sulfate reducing bacteria. The abundance of sequences for Thermodesulfovibro-like
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organisms and higher sulfate reduction rates at elevated temperatures, suggests that
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Thermodesulfovibro-like organisms may play a role in sulfate reduction in warmer
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environments. The rates of sulfate reduction presented here suggest that - within anaerobic
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niches of hydrothermal deposits - heterotrophic sulfate reduction may be quite common and
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can contribute to secondary productivity, underscoring the potential role of this process in both
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sulfur and carbon cycling at vents.
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Keywords: Hydrothermal Vent/Microbial Ecology/Primary productivity/Sulfate Reduction
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Subject Category: Geomicrobiology and microbial contributions to geochemical cycles
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INTRODUCTION
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Deep sea hydrothermal vent ecosystems are complex dynamic habitats characterized by
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steep gradients in temperature and geochemistry (Jannasch & Mottl, 1985). In these habitats,
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as hot hydrothermal fluid mixes with cold seawater, the precipitation of minerals creates large
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and complex hydrothermal chimney deposits. Within these permeable mineral structures, the
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continued mixing of chemically reduced, vent-derived fluids and oxidized seawater provides
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favorable conditions that support the growth of endolithic microbial communities (Schrenk et
61
al. 2003)
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Sulfide oxidation is considered to be one of the most important microbial
63
chemosynthetic pathways at ridge ecosystems, as evidenced by the ubiquity of sulfide oxidizing
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Epsilon- and Gammaproteobacteria at ridge environments (Nakagawa et al. 2004; Huber et al.
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2007; Nakagawa & Takai, 2008; Nakagawa et al. 2005; Campbell et al. 2006). To date,
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significantly less attention has been paid to the distribution and magnitude of sulfate reduction
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at vents, though sulfate reducing bacteria and archaea have frequently been isolated from deep
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sea hydrothermal environments (Houghton et al. 2007; Audiffrin et al. 2003; Alazard et al.
69
2003; Jannasch et al. 1988; Blöchl et al. 1997). Moreover, analyses of functional genes that
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express key proteins required for sulfate reduction suggest there is a high diversity of sulfate
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reducing organisms at vents, higher than predicted via 16S rRNA gene analyses alone
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(Nakagawa et al. 2004; Nercessian et al. 2005).
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From a biogeochemical and bioenergetic perspective, both sulfide oxidation and sulfate
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reduction would be favored at hydrothermal vents, though to varying degrees as a function of
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environmental chemistry. Sulfide oxidation is most favorable when coupled to oxygen or nitrate
3
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as an electron acceptor (Amend & Shock 2001). Around vents, sulfide is typically in µM to mM
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concentrations (Butterfield, et al. 1994; Butterfield et al. 1994), while oxygen and nitrate are
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around 110 and 40 µM respectively (Johnson et al. 1986). In contrast, sulfate reduction is highly
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favored in anoxic niches at vents, as it is in other marine anaerobic environments (Muyzer &
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Stams, 2008). Here, as in most marine systems, sulfate is abundant at 28 mM two to three
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orders of magnitude higher than oxygen At vents, sulfate reduction would occur in regimes
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where seawater-derived sulfate is still present but oxygen is absent, e.g. within hydrothermal
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vent deposits. Sulfate reducing microorganisms commonly use hydrogen and/or dissolved
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organic matter as electron donors, both of which are found within hydrothermal fluids (Lang et
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al. 2006; Cruse & Seewald, 2006). Sulfate reduction –as a function of its extent and magnitude-
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could readily influence the cycling of sulfur and sulfur isotopes, as well as carbon, within
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hydrothermal environments.
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To date, studies have quantified rates of sulfate reduction in hydrothermal-influenced
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sediments (Weber & Jorgensen, 2002; Jorgensen et al. 1992; Elsgaard et al. 1994; Kallmeyer &
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Boetius, 2004; Elsgaard et al. 1994; Elsgaard et al. 1995) and isolated vent microorganisms
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(Hoek et al. 2003). In contrast to the numerous studies of sulfate reduction in marine sediments
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(Canfield 1989), studies of sulfate reduction in hydrothermal deposits are few (Bonch-
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Osmolovskaya et al. 2011), due in part to the challenges associated with sampling and studying
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the heterogeneous and consolidated sulfide deposits typical of hydrothermal vent chimneys.
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Here we present rates of microbially mediated sulfate reduction from three distinct,
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active hydrothermal “chimneys” found in the Middle Valley field along the Juan de Fuca Ridge,
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as well as assessments of bacterial and archaeal diversity, estimates of total biomass and the
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abundance of functional genes related to sulfate reduction, and in situ geochemistry. These
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analyses further our understanding of sulfate reduction (including rates, diversity and
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distribution of known sulfate-reducing microbes) in vent ecosystems. Moreover, they
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underscore the potential role of heterotrophic sulfate reduction in hydrothermal systems, and
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constrain their potential influence on both sulfur and carbon cycling,
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METHODS
Geologic Setting and Sampling of hydrothermal deposits
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Middle Valley (48°27’N, 128°59’ W) is an intermediate spreading, axial rift valley,
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located along the Endeavor Segment of the Juan de Fuca Ridge in the Northwest Pacific ocean.
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Layers of continental-derived sediments characteristically cover Middle Valley, though the
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hydrothermal vents remain prominent above the sediments. Hydrothermal deposits were
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collected from 3 active hydrothermal spires during dive 4625 with the HOV Alvin (R/V Atlantis
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expedition AT15-67, July 2010) and brought to the surface in a sealed, temperature-insulated
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polyethylene box. Samples were recovered from actively venting sulfide deposits at Needles
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(48.45778, -128.709, 2412.212 m, Tmax=123°C), Dead Dog (48.45603,-128.71, 2405.268 m,
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Tmax=261°C), and Chowder Hill (48.455543, -128.709, 2398.257 m, Tmax =261°C) vents. Once on
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board ship, samples were directly transferred to sterile anaerobic seawater and
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handled/processed using appropriate sterile microbiological techniques. Subsamples were
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immediately transferred to gastight jars (Freund Container Inc.), filled with sterile anaerobic
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seawater containing 2 mM sodium sulfide at pH 6, and stored at 4°C. Upon return to the
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laboratory, all samples were provided with fresh 2 mM sulfidic, anaerobic seawater every 8 to
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12 weeks and were kept in the dark and 4°C prior to incubation.
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Vent fluid volatile geochemistry via in situ mass spectrometry
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In situ concentration of dissolved volatiles (H2S, H2, CO2, O2, and others.) were measured
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at each site with an in situ mass spectrometer (ISMS) as previously described (Wankel et al.
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2011). Briefly, dissolved volatiles were quantified in situ by sampling vent effluent for up to 10
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minutes, until partial pressures reached steady state (data was monitored in real time within
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the submersible). Concentrations were determined from empirically derived calibrations and
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validated by comparison with discrete samples collected using titanium gastight samplers.
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Measuring sulfate reduction rates
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Hydrothermal deposits were homogenized in a commercial blender (Xtreme™ blender,
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Waring Inc.) under a nitrogen atmosphere. Anaerobic homogenization was designed to
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minimize fine-scale geochemical and microbial heterogeneity and facilitate more accurate
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experimental replication. Hydrothermal homogenate (made up of both mineral deposit and
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interstitial fluid)) was aliquoted volumetrically (7.5 mL, ca. 29 g wet weight and ca. 20 g dry
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weight) into Balch tubes in an anaerobic chamber. The tubes were supplemented with 15 mL of
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sterile artificial vent fluid media designed to mimic the geochemical conditions within a sulfide
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deposit (pH 6, 14 mM SO42-, 2.3 mM NaHCO3, 1 mM H2S, and 10 μM each of pyruvate, citrate,
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formate, acetate, lactate). Organic acid concentrations are comparable to those measured in
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situ (Lang et al. 2006). Sufficient 35SO42- was added to achieve 555 kBq (15 μCi) of activity. Due
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to technical difficulties with post processing methodology, shipboard incubations using fresh
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material were not successful. The data presented here were generated using samples that had
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been kept at 4 °C and refreshed with vent-like effluent (as described above) for one year.
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Samples were incubated anaerobically for 7 days at 4, 30, 40, 50, 60, 80 and 90°C. Controls for
6
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sulfate reduction consisted of samples amended with 28 mM molybdate, a competitive
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inhibitor of sulfate reduction (Saleh et al. 1964; Newport & Nedwell, 1988). Six biological
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replicates were run for each treatment, and three biological replicates for each control. Upon
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completion, reactions were quenched with the injection of 5 mL 25% zinc acetate (which is ~20-
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fold more Zinc than the maximum sulfide concentration), and all samples were frozen at -20° C
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for further analysis.
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To determine sulfate reduction rates, samples were thawed and the supernatant was
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removed and filtered through a 0.2 μm syringe filter. The crushed deposits that remained in the
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tube were washed three times with deionized water to remove any remaining sulfate. One
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gram (wet weight) of crushed deposit was analyzed via chromium distillation (see Supplemental
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Methods) and sulfate reduction rates (SRR) were calculated as in (Fossing & Jorgensen,
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1989)using the following calculation.
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SRR =
Eq.1
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Where nSO42- is the quantity (in moles) of sulfate added to each incubation (14 mM * 15 mL =
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210 μmol), a is the activity (dpm) of the trapped sulfide, 1.06 is the fractionation factor
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between the sulfide and sulfate pools, A is the activity of the sulfate pool at the completion of
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the incubation and t is the incubation time (days). The rates are presented in units of nmol S g -1
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day-1.
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DNA Extraction
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Immediately prior to conducting the rate experiments, a subsample of homogenized
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hydrothermal deposit was removed and frozen at -80° C for molecular analysis. DNA was
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extracted from this crushed deposit sample with a protocol modified from (Santelli et al. 2008).
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Subsamples were washed with 0.1 N HCl, followed by two rinses with a sterile solution
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containing 10 mM Tris (pH 8.0) and 50 mM EDTA. A known mass of material was added to
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PowerSoil beadbeating tubes(MoBio Laboratories, Carlsbad CA), incubated at 70°C for 10
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minutes, and then amended with 200 ng of poly-A. Subsamples were subjected to beadbeating,
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followed by three cycles of freeze-thaw steps to further lyse cells. Nucleic acids were extracted
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using hot phenol (60°C for 3 min.), followed by two chloroform:isoamyl separations and
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precipitated with ethanol. DNA was resuspended in TE (pH 8.0) and quantified using the Qubit™
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fluorometer (Life Technologies, Grand Island, NY).
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Enumeration of gene abundance via quantitative PCR
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Quantitative PCR (qPCR) was used to determine the abundance of bacterial and archaeal
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16S rRNA genes. In addition, qPCR was used to enumerate the abundance of sulfate reducing
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prokaryotes by amplifying the adenosine 5‘-phosphosulfate reductase (aprA) gene with primers
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targeting sulfate reducing bacteria and archaea (Christophersen et al. 2011). Primers specific to
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bacterial dissimilatory sulfite reductase (dsrA) (Kondo et al. 2004) and Deltaproteobacteria 16S
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rRNA genes (Stults et al. 2001) provide alternate estimates of sulfate reducing bacteria
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populations. Quantification was performed in triplicate with the Stratagene MX3005p qPCR
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System (Agilent Technologies) using the Perfecta SYBR FastMix with low ROX (20 µL reactions,
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Quanta Biosciences, Gaitherburg, MD), specific primers and annealing temperatures (Table 1)
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and 10 ng of template gDNA. The temperature program for all assays was 94°C for 10 minutes,
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35 cycles of 94°C for 1 minute, the annealing temperature for 1 minute (Table 1), extension at
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72°C for 30 seconds and fluorescence read after 10 seconds at 80°C. Following amplification,
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dissociation curves were determined across a temperature range of 55°C to 95°C. Ct values for
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each well were calculated using the manufacturer’s software. Plasmids containing bacterial and
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archaeal 16S rRNA and functional gene inserts (amplified from Arcobacter nitrofigulis (ATCC
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33309), Methanosarcina acidovorans and Desulfovibrio vulgaris Hildenborough (ATCC 29579/
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NCIMB 8303/ AE017285) respectively) were used as standards for calibration (see
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Supplemental Methods for more detail).
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Sequencing and Phylogenetic Analysis via 454 pyrosequencing
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DNA samples were sequenced by Research and Testing Laboratory (Lubbock, TX) using 454
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pyrotag methods similar to those described previously (Dowd et al. 2008). All samples were
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sequenced using a 454FLX instrument (Roche Inc.) with Titanium™ reagents. The resulting
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bacterial and archaeal 16S rRNA and drsB genes (primers in Table 1) datasets were analyzed via
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Mothur (Schloss et al. 2009). Sequences were trimmed, quality checked, aligned to the SILVA-
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compatible alignment database reference alignment (dsrB gene datasets were aligned to a dsrB
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gene database generated from the Ribosomal Database Project (RDP)), analyzed for chimeras,
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classified against the Greengenes99 database and clustered in to OTUs (see Supplemental
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Methods for more detail). Rarefaction curves were used to examine the number of OTUs as a
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function of sampling depth. Alpha diversity was assessed by generating values from the Chao1
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richness estimators and the inverse Simpson diversity index.
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Sequence Accession numbers
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The 16S rRNA and dsrB gene sequences reported in this study have been submitted to
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Sequence Read Archive under the accession numbers SRX154520 through SRX154528.
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RESULTS
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Physical and geochemical characteristics of the study sites
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The hydrothermal deposits sampled from Middle Valley were all relatively friable and
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were composed predominantly of anhydrite (CaSO4, M. Tivey, pers. comm). Chowder Hill and
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Dead Dog had the highest observed venting fluid temperatures (measured in situ at 261°C),
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followed by Needles (123°C). In situ measurements of dissolved hydrogen sulfide (H2S) revealed
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significant differences in hydrothermal fluid composition among hydrothermal deposits.
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Unfortunately the inline pH probe with the ISMS malfunctioned during the dive. Using
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previously reported pH values (Butterfield et al. 1994), Chowder Hill would have the highest in
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situ measurement of total sulfide (3.9 mM), followed by Dead Dog (2.2 mM), and Needles (0.59
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mM) (Table 2). These concentrations are within the same magnitude of previously reported H2S
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in focused vent fluids at Middle Valley (Butterfield et al. 1994). Chowder Hill did exhibit the
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highest in situ concentration of hydrogen (1.86 mM) followed by Dead Dog (1.66 mM) and
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Needles (~1.42 mM). These values are also consistent with previous studies (Cruse & Seewald
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2006), as well as gastight samples collected and analyzed shipboard (M. Lilley, pers. comm).
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Sulfate Reduction Rates
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Among all samples, sulfate reduction was observed at temperatures between 4°C to
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90°C (Figure 1). Maximal rates of sulfate reduction were observed between 88-90°C (2670 nmol
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g-1 day-1 at Needles, 1090 nmol g-1 day-1 at Chowder Hill, and 142 nmol g-1 day-1 at Dead Dog;
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Figure 1). Notably, the highest sulfate reduction rates were observed from Needles samples,
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which were ~20-fold higher than those observed at Dead Dog, and ~2-fold greater than at
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Chowder Hill. Many of the rates exhibit large deviations due to the high variability among the
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biological replicates, most likely due to persistent mineralogical and microbiological
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heterogeneity across incubations, even after homogenization. Sulfate reduction was also
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observed in molybdate amended experiments, though we suspect that molybdate was
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scavenged by minerals that attenuated the effect of the inhibitor as has been previously
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observed in metal-rich environments (Bostick et al. 2003; Xu et al. 2006).
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Quantification of Taxonomic and Functional Genes
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The abundance of total bacteria, archaea (16S rRNA genes), sulfate reducing
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prokaryotes (aprA gene) and sulfate reducing bacteria (dsrA and Deltaproteobacteria specific
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16S rRNA genes) were investigated in each deposit by quantitative PCR (Figure 2). Microbial
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density (as estimated by 16S rRNA gene copies g-1 mineral) was greatest at Needles and lowest
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at Dead Dog. Microbial communities at each site were dominated by archaea (Figure 2A), with
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Needles showing the highest ratio of archaea to bacteria (227:1 as compared to 14:1 at Dead
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Dog or 17.5:1 at Chowder Hill). Assuming an average of 4.19 copies of 16S rRNA gene per
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bacterium and 1.71 copies of 16S rRNA gene per archaeon genome (Lee et al. 2009;
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Klappenbach et al. 2001), Needles hosts a microbial community of 4.12 x 108 cells g-1 sample, 3
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orders of magnitude higher than Chowder Hill (8.96 x 105 cells g-1 sample) and Dead Dog (5.65 x
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105 cells g-1 sample).
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16S rRNA gene primers specifically targeting ribotypes allied to Desulfovibrio,
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Desulfomicrobium,
Desulfuromusa,
and
Desulfuromonas
were
used
to
enumerate
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Deltaproteobacteria known to mediate sulfate reduction in many marine systems (Stults et al.
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2001). However, given the difficulty in amplifying 16S rRNA genes from deep-sea thermophiles
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with typical primer sets - due to mismatches with limited sequence representation in GenBank -
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it is possible that these assays similarly underestimate abundances in these environments
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(Teske & Sorensen, 2008). Deltaproteobacterial abundance at Needles was approximately 4.48
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x 105 copies g-1 sample (approximately 26% of the entire bacterial population), though none
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were detected at Chowder Hill or Dead Dog (data not shown). The abundance of both
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functional genes for sulfate reduction, dsrA and aprA, was greatest at Needles and lowest at
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Dead Dog (a pattern similar to that seen in the 16S rRNA gene abundance estimates; Figure 2B).
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If we assume an average of 1 dsrA gene copy per genome (Klein et al. 2001; Kondo et al. 2004),
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the proportion of sulfate reducing bacteria in the bacterial populations is only 2.7% in Needles
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as compared with 28% in Dead Dog and 53% at Chowder Hill.
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Microbial Diversity
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454 pyrotag sequencing (bacterial V1-V3 and archaeal V3-V4 of the 16S rRNA gene),
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rarefaction analyses, and diversity metrics all revealed measureable differences in microbial
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community composition among the three hydrothermal deposits (Figure 3, Figure 4, Table 3).
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Via these assessments, Needles hosts the least diverse assemblage of bacteria and archaea,
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while Chowder Hill and Dead Dog host communities of comparable diversity. Examination of
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OTUs at 97%, 95% and 92% sequence similarity further reveal differences in microbial
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community membership among the three sites. Among archaea at the 97% level, only two
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archaeal OTUs (1% of all archaeal OTUs classified) are shared among the hydrothermal
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deposits. The sequences classified to these OTUs represent 69%, 48% and 18% of all the library
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sequences from Needles, Dead Dog and Chowder Hill respectively. One of these OTUs is allied
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to the ammonium oxidizing archaeal Candidatus Cenarchaeum in the phylum Thaumarchaeota,
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and accounts for 35% of Needles and less than 5.0% of Dead Dog or Chowder Hill library
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sequences. The other OTU is allied to a thermophilic sulfur respiring archaeon within the class
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Thermoplasmata. Nearly 40% of the archaeal sequences from Dead Dog were allied to this
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archaeon. Methanogens allied to Methanocaldococus comprised about 1.0% of the total
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archaeal sequences from Dead Dog, and were not represented in the libraries from Chowder
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Hill or Needles. Most of the archaeal diversity at Chowder Hill (80% of sequences) and Dead
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Dog (50% of sequences) was unclassified. No sequences allied to true sulfate reducing archaeal
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lineages such as Archaeoglobus fugilis or Aciduliprofundus boonei were recovered. However,
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the potential diversity of thermophilic sulfate reducing archaea in these samples is likely much
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greater than suggested here. This may be explained in part by biases underlying DNA
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extractions, primer binding and sequencing. For example the archaeal sequencing primers used
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in this study only target about one third (34%, as assayed by Probe Match; Cole et al. 2005) of
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the Archaeoglous-like sequences contained in the RDP database. Furthermore, the primers may
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miss members of the dominant Thermoplasmatales as in silico analysis only returns 48%
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(1715/3558 sequences) of the RDP reported sequences. In total, these archaeal sequencing
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primers (349F-806R) miss 42% of the total archaeal sequences (67713/117373 sequences) in
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the RDP database. Similar bias has been reported in other studies in the deep sea and deep
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subsurface biotopes (e.g. Dhillion et al., 2003, 2005, Teske et al. 2007).
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Among bacteria at the 97% similarity level, 54 of the bacterial OTUs classified (7.0%)
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were shared among all hydrothermal deposits, and account for 84%, 80%, 71% of the
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sequences from Chowder Hill, Needles and Dead Dog respectively (Figure4). One of these OTUs
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accounted for 44%, 36% and 25% of the sequences from Chowder Hill, Dead Dog and Needles
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respectively. Aligning representative sequences from this OTU via Blast-n (Altschul et al. 1997)
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reveals a best match to Thermodesulfovibrio, an anaerobic, thermophilic, sulfate-reducing
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bacterium, from the phylum Nitrospira (81% identity). Given its abundance, we postulate that it
300
likely contributes substantially to the high thermophilic sulfate reduction rates. Furthermore,
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the
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Thermodesulfovibrio (Supplemental Table S1). Other dominant groups of bacteria include
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members of the
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proteobacteria. Via Blast-n, most of the unclassified sequences matched to partial 16S rRNA
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gene sequences from hydrothermal vent fluid communities (Nunoura et al. 2010; Sylvan et al.
306
2012). Sequences classified as Deltaproteobacteria comprised 5.5%, 8.2%, or 14%, of the total
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population of Chowder Hill, Needles, or Dead Dog respectively. While Dead Dog may have had
308
the highest proportion of its amplicons classified as Deltaproteobacteria, sequences related to
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known sulfate reducers within the Deltaproteobacteria (Desulfobacteraceae, Desulfobulbus
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rhabdoformis, Desulfovibrio) were only found at Needles and comprised 1.1% of the 16S rRNA
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gene library. The majority of the sequences classified as Deltaproteobacteria in each of the
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three sites were from one unclassified Deltaproteobacterial OTU consisting of 4.4, 5.3, and 13%
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of the sequences from Chowder Hill, Needles and Dead Dog respectively.
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DISCUSSION
dsrB
gene
library
was
dominated
Gammaproteobacteria,
by
sequences
Bacteroidetes,
phylogenetically
Deltaproteobacteria
allied
and
to
α-
315
Sulfate reduction rates measured in deposits recovered from the Middle Valley vent
316
field reveal the potential for active sulfate reduction within hydrothermal deposits. The
317
magnitude of all measured rates (from 15.7 nmol g-1 day-1 at Dead Dog at 60°C to 2670 nmol g-1
318
day-1 at Needles at 90°C) under experimental conditions were markedly higher than those
319
typically observed in non hydrothermal deep sea sediments (0.1-10 nmol g-1 day-1, converted
320
here for comparison assuming an average sediment density of 2 g cm-3; Elsgaard, Isaksen, et al.
14
321
1994; Weber & Jorgensen, 2002; Joye et al. 2004). These rates are comparable in magnitude to
322
those previously observed in hydrothermally influenced sediments (eg. Guaymas basin or Lake
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Tanganyika (Weber & Jorgensen, 2002; Kallmeyer & Boetius, 2004; Elsgaard, Isaksen, et al.
324
1994; Elsgaard, Prieur, et al. 1994), even though the availability of organic carbon is markedly
325
higher in these hydrothermal vent sediments, with Guaymas having up to 200-times greater
326
concentrations of organic carbon (Lang et al. 2006; Cruse & Seewald, 2006; Chen et al. 1993).
327
To date, the only other measurement of sulfate reduction from sulfide deposits along the East
328
Pacific Rise exhibited rates comparable to those reported here, (Bonch-Osmolovskaya et al.
329
2011), but it should be noted that their samples were incubated under pure H2 atmosphere.
330
Notably, the maximum rates of sulfate reduction in Middle Valley sulfides occurred at
331
90°C in all three deposits. This is in contrast to measurements of sulfate reduction in
332
hydrothermal sediments, where the greatest rates are often observed between 40-70°C, and
333
more modest rates of sulfate reduction have been reported between 80-91°C (Weber &
334
Jorgensen, 2002; Elsgaard, Prieur, et al. 1994; Elsgaard, Isaksen, et al. 1994). The relatively low
335
or insignificant sulfate reduction rates between 4-80°C suggest Middle Valley deposits harbor a
336
high proportion of hyperthermophilic sulfate-reducing microbes.
337
The significant differences in the rates we observed among deposits (Kruskal-Wallis,
338
p<0.0001) are likely due to differences in biomass and the composition of microbial
339
communities that are influenced by the geochemistry of each deposit. Indeed, microbial
340
biomass (as estimated by 16S rRNA genes) directly correlates to rates of activity and is likely
341
one of the strongest factors affecting the observed rates of sulfate reduction (Pearson
342
correlation coefficient r=0.879, p<0.0005). Needles had both the highest observed rates as well
15
343
as the highest cell density (Figure 2 and 3). Of all the deposits sampled, Needles had the lowest
344
venting fluid temperature (123°C) resulting in the largest zone of microbial habitability.
345
Consistent with this, Needles also had the greatest abundance of dsrA and aprA genes per
346
gram, suggesting a larger potential sulfate reducing community. Here, Deltaproteobacteria
347
allied to Desulfovibrio, Desulfobulbus, Desulfobacteria, and Desulfuromonas account for 25.7%
348
of the bacterial community. These clades of Deltaproteobacteria were not observed at
349
Chowder Hill or Dead Dog by either qPCR enumeration or pyrosequencing. Cultured
350
representatives from some of these Deltaproteobacterial clades (Desulfovibrio vulgaris and
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Desulfovibrio desulfuricans) have been shown to reduce sulfate at high rates (ranging from 10-
352
1340 nmol min-1 mg-1 protein) with varying electron donors (Fitz & Cypionka, 1991; Cypionka &
353
Konstanz, 1989).
354
Thermodesulfovibrio-like organisms dominated the bacterial communities within each
355
hydrothermal deposit (35-44%; Fig. 4). Thermodesulfovibrio sp. are considered obligately
356
anaerobic, thermophilic bacteria that can reduce sulfate and other sulfur compounds (Garrity &
357
Holt, 2001). In pure cultures, members of this genus are able to link growth with hydrogen and
358
a limited range of organic carbon molecules (formate, pyruvate and lactate), maintaining
359
optimal growth between 55-70°C (Sekiguchi et al., 2008). Needles has a greater proportion of
360
sequences (from pyrosequencing) related to Thermodesulfovibrio-like species than the other
361
two deposits. The combination of many sequences related to a thermophilic sulfate reducing
362
bacteria and higher rates of sulfate reduction at elevated temperatures and the, suggests that
363
Thermodesulfovibrio-like organisms may play a role in sulfate reduction in warmer
364
environments. However, constraining the relative proportion of sulfate reduction by
16
365
Thermodesulfovibrio-like organisms in these mixed communities was beyond the scope of this
366
study.
367
It is unclear why Chowder Hill and Dead Dog exhibit large differences in rates of sulfate
368
reduction despite other similarities in geochemistry and biomass. One plausible explanation
369
might be that different types of biological interactions (e.g. syntrophy or competition) occur
370
due to differences in the composition and distribution of microbial communities within the
371
mineral matrix of each deposit. Slight differences in community composition, like Dead Dog
372
having a higher representation of sequences related to sulfur respiring (Thermoplasmata) and
373
methanogenic (Methanocaldococus-like) archaea than Chowder Hill, may lead to biological
374
interactions that have different implications for rates of sulfate reduction in each deposit. Also,
375
substrate competition for H2 or consumption of locally produced DOC (Oremland & Polcin,
376
1982; Lovley & Phillips, 1987) may be more prevalent in one deposit over another. Future
377
experiments should aim to better resolve how specific interaction between populations, for
378
example, syntrophy or competition for a common substrate, may influence sulfate reduction.
379
The relevance of heterotrophic sulfate reduction on hydrothermal vent biomass production
380
and biogeochemistry
381
Heterotrophic sulfate reduction is likely a prominent metabolic mode within Middle
382
Valley sulfides and sediments, and the sulfate reduction rate data herein (which solely measure
383
hetrotrophic sulfate reduction) support that supposition. Sedimented vent fields typically
384
contain allochthonous organic carbon that could readily support heterotrophy. Indeed, at
385
Middle Valley, bottom waters contain 3.5 mg DOC/L (about 7 fold higher than the overlying
386
surface seawater), while porewater concentrations range from 0.1 – 84.0 mg DOC/L at
17
387
sediment depths to 200 mbsf (Ran & Simoneit, 1994). Based on data from culture studies of
388
Desulfovibrio strains, including the H+/H2 ratio of 1.0 for Desulfovibrio vulgarius Marburg (Fitz &
389
Cypionka, 1991), the P/2e- ratio (number of ATPs produced for every 2 electrons transferred to
390
an electron acceptor) of 1/3 for Desulfovibrio gigas (Barton et al. 1983), and the assumption
391
that 10% of ATP production supports growth (20 mmol ATP per gram biomass), our estimates
392
suggest that - at our maximum empirically measured rates - heterotrophic sulfate reduction
393
could support 140 g biomass yr-1 (~1.5 x 1014 cells) at Chowder Hill (volume = 109,900 cm3), 16 g
394
biomass yr-1 (~1.7 x 1013 cells) at Needles (volume = 5495 cm3), and 2.1 g biomass yr-1 (~2.2 x
395
1012 cells) at Dead Dog (volume=12560 cm3). While these values may be small in comparison to
396
global estimates of chemoautotrophic biomass production on the global ridge system (1010 -
397
1013 g of biomass yr-1 ;McCollom & Shock 1997; Bach & Edwards 2003), the sulfide produced by
398
these heterotrophic sulfate reducers could represent up to 3% of the H 2S flux from Middle
399
Valley deposits (given previously published vent fluid flow rates from the Main Endeavor field,
400
(Wankel et al. 2011)). Additional rate measurements that represent the diversity of physico-
401
chemical conditions found within deposits or ridge systems are necessary to better constrain
402
the contribution of heterotrophic sulfate reducers to global vent biomass and geochemistry.
403
Hydrothermal vents are dynamic environments where carbon and sulfur cycling are
404
intimately linked. Both autotrophic and heterotrophic sulfate reducing microbes have been
405
isolated from vents, and the data shown here are among the first to constrain the potential for
406
heterotorophic sulfate reduction at vents (in particular those with higher organic carbon loads),
407
as well as the relationship between sulfate reduction rates, temperature, microbial biomass
408
and community density and composition. These data, as well as the vent field estimates of
18
409
sulfate reduction, , underscore the relevance of sulfate reduction in hydrothermal ecosystems
410
and further indicate the need for continued studies of sulfur cycling along ridge systems.
411
412
ACKNOWLEDGMENTS:
413
We are grateful for the expert assistance of the R/V Atlantis crews and the pilots and
414
team of the DSV Alvin for enabling the collections of hydrothermal deposits used in our
415
experiments. We also thank Steve Sansone, Dr. Joseph Ring, Ms. Julie Hanlon, Dr. Kathleen
416
Scott, Dr. Vladimir Samarkin, Dr. David Johnston, and Dr. Jan Amend for providing assistance
417
with various technical aspects of the experiments. We are also very thankful for the
418
constructive feedback from the reviewers. Financial support for this research was provided by
419
the National Science Foundation (NSF OCE-0838107 and NSF OCE-1061934 to P.R. Girguis), and
420
the National Aeronautic and Space Administration (NASA-ASTEP NNX09AB78G to C. Scholin and
421
P. R. Girguis and NASA-ASTEP NNX07AV51G to A. Knoll and P. R.Girguis).
422
Supplementary information is available at ISME’s website
423
424
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